Enhancement of the catalytic activity of D-lactate dehydrogenase from Sporolactobacillus laevolacticus by site-directed mutagenesis

Article abstract of DOI:10.1016/j.bej.2018.02.015

Sporolactobacillus laevolacticus is a producer of D-lactic acid with high optical purity. The amino acid sequence of D-lactate dehydrogenase (D-LDH) from S. laevolacticus was compared with those of other lactate producers. To enhance the activity of D-LDH from S. laevolacticus, some amino acid residues close to the substrate binding site or the active center were replaced by site-directed mutagenesis. Ala234 of D-LDH from S. laevolacticus was found to be an important amino acid residue that positively affects catalytic activity. Site-saturation mutagenesis of the 234th residue was performed. The mutant D-LDH at the 234th residue from alanine to serine or glycine showed enhanced catalytic activity toward pyruvate. The kinetic analysis revealed that the k_cat/K_m of D-LDH_A234S and _A234G on pyruvate increased 1.9- and 1.2-fold, respectively. Furthermore, the double mutant D-LDH_T75L/A234S improved k_cat/K_m by 6.8-fold compared to that of wild-type D-LDH. These results showed the potential of the mutant D-LDH for microbial production of D-lactic acid by heterologous expression.

Full text of DOI:10.1016/j.bej.2018.02.015

Accepted Manuscript
Title: Enhancement of the catalytic activity of D-lactate
dehydrogenase from Sporolactobacillus laevolacticus by
site-directed mutagenesis
Authors: Kento Nakano, Shoichi Sawada, Ryosuke Yamada,
Takashi Mimitsuka, Hiroyasu Ogino
PII:
DOI:
Reference:
S1369-703X(18)30074-3
https://doi.org/10.1016/j.bej.2018.02.015
BEJ 6900
To appear in:
Biochemical Engineering Journal
Received date:
Revised date:
Accepted date:
6-11-2017
15-2-2018
19-2-2018
Please cite this article as: Kento Nakano, Shoichi Sawada, Ryosuke Yamada, Takashi
Mimitsuka, Hiroyasu Ogino, Enhancement of the catalytic activity of d-lactate
dehydrogenase from Sporolactobacillus laevolacticus by site-directed mutagenesis,
Biochemical Engineering Journal https://doi.org/10.1016/j.bej.2018.02.015
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Enhancement of the catalytic activity of D-lactate dehydrogenase from
Sporolactobacillus laevolacticus by site-directed mutagenesis
Kento Nakanoa, Shoichi Sawadaa, Ryosuke Yamadaa, Takashi Mimitsukab, and Hiroyasu
Oginoa,*
a Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho,
Naka-ku, Sakai, Osaka 599-8531, Japan.
b New Frontiers Research Laboratories, Toray Industries, Inc., 6-10-1 Tebiro, Kamakura,
Kanagawa 248-8555, Japan
*Corresponding author.
E-mail address: ogino@chemeng.osakafu-u.ac.jp (H. Ogino)
Graphical abstract
1

Highlights
 Finding of important amino acid residues that positively affect catalytic
activity.
 Optimization of an amino acid residue to enhance the activity of D-LDH.
 Site-directed mutagenesis improved the activity of D-LDH from S.
laevolacticus.
 Potential of the mutant D-LDH for microbial production of D-lactic acid.
ABSTRACT
Sporolactobacillus laevolacticus is a producer of D-lactic acid with high optical purity.
The amino acid sequence of D-lactate dehydrogenase (D-LDH) from S. laevolacticus
was compared with those of other lactate producers. To enhance the activity of D-LDH
from S. laevolacticus, some amino acid residues close to the substrate binding site or the
active center were replaced by site-directed mutagenesis. Ala234 of D-LDH from S.
laevolacticus was found to be an important amino acid residue that positively affects
catalytic activity. Site-saturation mutagenesis of the 234th residue was performed. The
mutant D-LDH at the 234th residue from alanine to serine or glycine showed enhanced
catalytic activity toward pyruvate. The kinetic analysis revealed that the kcat/Km of
2

D-LDH_A234S and _A234G on pyruvate increased 1.9- and 1.2-fold, respectively.
Furthermore, the double mutant D-LDH_T75L/A234S improved kcat/Km by 6.8-fold
compared to that of wild-type D-LDH. These results showed the potential of the mutant
D-LDH for microbial production of D-lactic acid by heterologous expression.
Keywords: D-lactate dehydrogenase, D-lactic acid, polylactic acid, site-directed
mutagenesis
3

1. Introduction
Lactic acid is a fermentation product required for the production of polylactic
acid (PLA) [1]. PLA is a sustainable alternative to petrochemical-derived plastics
because lactic acid can be produced on a large scale by the microbial fermentation of
biomass [2]. Moreover, PLA is a biodegradable and biocompatible material that can be
used in a wide variety of applications ranging from packaging to the production of
fibers and foams [3]. PLA is regarded as an environment-friendly alternative to
traditional plastics derived from fossil fuels [4].
PLA can exist in three stereochemical forms: poly (L-lactic acid) (PLLA), poly
(D-lactic acid) (PDLA), and poly (DL-lactic acid) (PDLLA); these are polymers of
L-lactic acid, D-lactic acid, and DL-lactic acid, respectively. The homopolymers, PLLA
and PDLA, have a melting point of approximately 170°C, while PDLLA depresses the
crystalline melting point to as low as 130°C [5, 6]. In addition, prior studies
demonstrated that blending PLLA and PDLA could create a stereocomplex PLA with a
melting point of 230°C, which is 50°C higher than those of the homopolymers [7, 8].
The fermentative production of L-lactic acid has advanced; however, the technology for
D-lactic acid remains immature [9, 10] and there exists an increasing demand for
4

optically pure D-lactic acid production.
Almost all lactic acid bacteria have both L-lactate dehydrogenase (L-LDH) and
D-lactate dehydrogenase (D-LDH). In lactic acid bacteria producing D-lactic acid, the
specific activity of purified D-LDH is much higher than that of purified L-LDH [11].
Sporolactobacillus laevolacticus (a D-lactic acid producer) is known to produce D-lactic
acid with relatively high optical purity [6, 12]. It was expected that as D-LDH of S.
laevolacticus had high catalytic activity, S. laevolacticus could produce D-lactic acid
with high optical purity and yield.
In this report, D-LDH from S. laevolacticus was overexpressed in Escherichia
coli, purified, and characterized in vitro. Enhancement of the specific activity of D-LDH
from S. laevolacticus was attempted by using site-directed mutagenesis. The mutation
points were identified by multiple sequence alignment of three LDHs. Furthermore,
site-saturation mutagenesis at the specific residue and combination of these mutations
were performed to construct mutant D-LDH that has superior catalytic activity.
5

2. Materials and methods
2.1. Construction of the expression plasmid
The D-LDH gene was amplified by PCR from pBL2 containing the gene
encoding D-LDH from S. laevolacticus JCM 2513 [13] using the forward primer
SLLDHD-FP2 (recognition site of Nde I underlined, 5′-GGA GAA TTC ATA TGA AAT
TCT TGA TGT ATG-3′) and the reverse primer SLLDHD-RP (recognition site of Xho I
underlined, 5′-TTG TCT CGA GTT TTG CCG ATA GCG GTT GTG CTT GAA CAA
G-3′). The PCR product was recovered by the High Pure PCR Product Purification Kit
(Roche Diagnostics, Indianapolis, IN, USA), and its size was confirmed as a 1,007-bp
band by 2% (w/v) agarose gel electrophoresis. The purified PCR product was digested
with Nde I and Xho I and ligated with the Nde I-Xho I-digested pET-21b(+) vector by
using Ligation high Ver. 2.0 (Toyobo, Osaka, Japan). The D-LDH gene was His-tagged
[hexa-histidine (6×His)] at the C-terminus and cloned downstream of the T7 promoter
in the pET-21b(+) expression vector. The recombinant plasmid DNA, pEDldh, was
transformed into both E. coli JM109 and E. coli BL21(DE3) cells for protein expression.
Mutant D-LDH genes were constructed using the QuikChange Site-Directed
6

Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and the
abovementioned recombinant plasmid vector, pEDldh, as a template. The recombinant
vector and all mutant genes were confirmed by DNA sequencing.
2.2. Expression and purification of D-LDHs
E. coli BL21(DE3) transformed with pEDldh and its derivatives, which
harbored the wild-type and mutant D-LDH genes, respectively, were cultured in
Luria-Bertani medium (10 g tryptone, 5 g yeast extract, 5 g NaCl) containing 100 mg/L
ampicillin sodium. A volume of 200 mL medium in a 500 mL baffled Erlenmeyer flask
was inoculated with the transformed cells and incubated at 37°C for 2-3 h with rotary
shaking at 150 rpm. When the optical density at 600 nm (OD600) reached 0.6–0.8, the
expression of D-LDHs was induced for 4 h at 25°C with shaking at 150 rpm by adding
isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM [14].
The cells were harvested by centrifugation at 7,150 × g for 5 min at 4°C and
re-suspended in a 50 mM Tris-HCl buffer (pH 7.0) twice. The suspension was disrupted
by intermittent ultrasonic disintegration at 80 W for 40 min in an ice bath. The
supernatant and precipitate of the resulting suspension were separated by centrifugation
7

at 11,200 × g for 5 min at 4°C into soluble and insoluble fractions, respectively.
Because the expressed D-LDHs were fused with a 6×His affinity purification
motif at their C-termini, affinity chromatography using the His-TrapFF column (GE
Healthcare, Little Chalfont, UK) was performed to purify the recombinant D-LDHs.
Supernatants containing the soluble recombinant D-LDHs were applied to a His-TrapFF
column, which was pre-equilibrated with an equilibration buffer (pH 7.0) containing 50
mM sodium phosphate and 10 mM imidazole at a flow rate of 1 mL/min. After the
His-TrapFF column was washed with 5 mL of washing buffer (pH 7.0) containing 50
mM sodium phosphate and 20 mM imidazole, the recombinant D-LDHs were finally
eluted with 2 mL of elution buffer (pH 7.0) containing 50 mM sodium phosphate and
200 mM imidazole. The 2 mL of eluate was packed into a cellulose tube and dialyzed
against 300 mL of 50 mM Tris-HCl buffer (pH 7.0) at 4°C overnight.
The protein concentrations were determined according to the Bradford method
using Protein Assay CBB Solution (Nacalai Tesque, Kyoto, Japan).
The expression and purity of the recombinant D-LDHs were monitored by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The
SDS-PAGE was performed by using stacking and separating gels containing 4% and
14.8% (w/v) acrylamide, respectively. The SDS treatment was performed by mixing 10
8

μL of samples and 10 μL of SDS sample buffer (pH 6.8) containing 62.5 mM Tris-HCl,
2% (w/v) SDS, 20% (v/v) glycerol, 2% (v/v) 2-mercaptoethanol, and 0.1% (w/v)
bromophenol blue and incubating at 95°C for 5 min.
2.3. D-LDH activity assay
The activity of D-LDHs was measured using sodium pyruvate and NADH. A
volume of 3 mL of the reaction mixture containing 10 mM sodium pyruvate and 0.2
mM NADH in 50 mM Tris-HCl buffer (pH 7.0) was pre-incubated at 27°C for 5 min.
To start the reaction, 2 μL of D-LDH solution was added. The NADH concentration was
continuously recorded by monitoring absorbance at 340 nm for 5 min during redox
reactions catalyzed by D-LDH. One unit of activity was defined as the amount of
enzyme that catalyzed the oxidation of 1 μmol NADH per minute at 27°C and pH 7.0.
2.4. Kinetic parameters of D-LDHs
A kinetic study of D-LDHs was performed using various concentrations of
sodium pyruvate. A volume of 3 mL of the reaction mixture containing 0.2, 0.5, 1, 5, 10,
9

20, 30, 60, 80 mM sodium pyruvate and 0.5 mM NADH in 50 mM Tris-HCl buffer (pH
7.0) was pre-incubated at 27°C for 5 min. To start the reaction, 5 μg of D-LDH was
added. The kinetic parameters (kcat/Km) were determined by Lineweaver–Burk plots. All
experimental data in this study were the average values of triplicate measurements.
10

3. Results and discussion
3.1. Multiple sequence alignment of D-LDHs
Lactic acid bacteria have both L-LDH and D-LDH. The specific activity of
D-LDH from a D-lactic acid producer is much higher than that of D-LDH from an
L-lactic acid producer [11]. To identify the target sites for site-directed mutagenesis,
amino acid sequence alignment of D-LDH from S. laevolacticus JCM 2513 (a D-lactic
acid producer) [13], Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 (a
D-lactic acid producer), and Lactobacillus casei ATCC 334 (an L-lactic acid producer)
was used as shown in Figure 1. Amino acid sequences of D-LDHs from L. bulgaricus
(accession number: CAI96942) and L. casei (accession number: YP_805451) were
retrieved
from
the
National
Center
for
Biotechnology
Information
(https://www.ncbi.nlm.nih.gov). Amino acid residues adjacent to the substrate binding
site or the active center, but not the active center, were selected as candidates. Ala234,
Thr260, Asn263, and Val296 of D-LDH from S. laevolacticus were different from those
from L. bulgaricus and conserved in those from L. casei compared with corresponding
residues in amino acid type. Especially, Thr260 of D-LDH from S. laevolacticus
11

corresponds to Val261 of D-LDH of L. bulgaricus, which is located near the catalytic
active center [15]. The 263th residue of D-LDH from S. laevolacticus is amino acid with
a hydrophilic side chain, Asn and Tyr in D-LDHs from S. laevolacticus and L. casei, a
L-lactic acid producer, respectively, while Gly in D-LDH from L. bulgaricus, D-lactic
acid producer. The 296th residue is amino acid with a hydrophobic side chain, Val and
Ile in D-LDHs from S. laevolacticus and L. casei, respectively, while Thr with a
hydrophilic side chain in D-LDH from L. bulgaricus. Therefore, it was assumed that the
replacements of Asn263 and Val296 of D-LDH from S. laevolacticus by Gly and Thr
would affect the catalytic activity. Thr75, Thr77, Ala78, Ala100, Gly297, and Phe299 of
D-LDH from S. laevolacticus were different from those from both L. bulgaricus and L.
casei. Amino acid residues of D-LDH from S. laevolacticus, which were same or similar
to those from L. casei and different from those from L. bulgaricus, were replaced to
match those found in D-LDH from L. bulgaricus. Variants of D-LDH_A234S, _T260V,
_N263G, _V296T, _T75L, _T77N, _A78V, _A100V, _G297A, and _F299Y of S.
laevolacticus were therefore constructed by site-directed mutagenesis. In Fig. 2, the
replaced amino acid residues were simply mapped onto the three-dimensional structure
of D-LDH from S. inulinus (PDB ID 4XKJ) that is 49% identical to that from S.
laevolacticus.
12

3.2. Heterologous expression of wild-type D-LDH of S. laevolacticus in E. coli
The wild-type D-LDH gene of S. laevolacticus was cloned into the expression
vector pET-21b(+), and 6×His-tagged wild-type D-LDH was overexpressed in E. coli
BL21(DE3) under the control of the T7 promoter. Protein expression was induced with
1.0 mM IPTG, and the expressed proteins were analyzed by SDS-PAGE (Fig. 3). Most
of the expressed 6×His-tagged wild-type D-LDH was found in a soluble fraction. After
purification by affinity chromatography with the His-TrapFF column and dialysis, a
single band of approximately 36 kDa appeared, confirming the calculated molecular
mass of 38 kDa derived from the amino acid sequence of a single subunit. The yield of
6×His-tagged wild-type D-LDH purified from the cell disruption supernatant was 39.6%.
The specific activity of purified 6×His-tagged wild-type D-LDH was calculated to be
24.0 U/mg in the reduction of sodium pyruvate with NADH as a cofactor at 27°C.
Remaining activities of the wild-type D-LDH after 15 min-incubation at
various temperatures are showed in Fig. 4. The wild-type D-LDH was stable under
30ºC.
13

3.3. Characterization of specific activities of mutant D-LDHs
Most 6×His-tagged mutant D-LDHs (D-LDH_T75L, _A78V, _A100V, _A234S,
_T260V, _N263G, _V296T, _G297A, and _F299Y) were expressed in the soluble
fraction, while D-LDH_T77N was expressed in the insoluble fraction. After the
purification and measurement of activity, the expressed 6×His-tagged mutant D-LDHs
except for D-LDH_T77N were found to have catalytic activity. Figure 5 shows the
specific activities of mutant D-LDHs, except for those of D-LDH_T77N.
The specific activities of D-LDH_A78V, _T260V, and _F299Y were 23.0 ± 0.6,
23.1 ± 3.7, and 25.3 ± 2.3 U/mg, respectively, which are similar to that of the wild-type
D-LDH. The D-LDH_T75L and _A234S showed higher catalytic efficiency than the
wild-type D-LDH. The specific activities of D-LDH_T75L and _A234S were 37.7 ± 1.1
and 40.7 ± 0.9 U/mg, respectively.
3.4. Site-saturation mutagenesis of residue 234 of D-LDH
In D-LDH from S. laevolacticus, Ala234 neighbors the active center Arg235.
The D-LDH_A234S showed the highest specific activity among the single mutant
14

D-LDHs. This suggested that 234th amino acid residue played an important role in
catalytic reaction. Site-saturation mutagenesis of residue 234 was subsequently
performed. Although D-LDH_A234P was expressed in the insoluble fraction, mutant
D-LDHs, except for D-LDH_A234P, were expressed in the soluble fraction. The
site-saturation mutagenesis at position 234 revealed that only D-LDH_A234S, _A234G,
_A234C, and _A234T retained catalytic activity. Figure 5 shows the results of these
mutant D-LDH activity assays. D-LDH mutants, in which the 234th residue was changed
from Ala to Cys or Thr, showed lower catalytic activity than the wild-type D-LDH. In
contrast, D-LDH_A234S and D-LDH_A234G respectively demonstrated 1.7-fold and
1.6-fold improvement in specific activity over the wild-type D-LDH (40.7 ± 0.9 and
38.0 ± 2.6 U/mg, respectively). The capacity of the substrate binding pocket depends
greatly on the size of the side chain [16]. Because the size of Ser is similar to that of Ala
and Gly is the smallest residue, the replacement of Ala234 by a smaller amino acid
significantly increased the specific activity. Thus, the catalytic activity was lost because
of mutation at position 234 of D-LDH to amino acids larger than Thr because of the
steric constraint, and the catalytic activity increased with decreasing sizes of the amino
acid residues. According to the D-LDH from S. inulinus (PDB ID 4XKJ), Ser233
(Ala234 in that from S. laevolacticus) forms hydrogen bonds to the conserved Asn231
15

(Asn232 in that from S. laevolacticus). The possible hypothesis is that mutation from
Ala to Ser at position 234 of D-LDH from S. laevolacticus may have affected
conformation of substrate binding site.
Since two mutations with improved activity were identified, the double mutant
D-LDH_T75L/A234S and _T75L/A234G were created by site-directed mutagenesis.
The specific activity of the double mutant D-LDH_T75L/A234S and _T75L/A234G
were improved to 66.8 ± 2.2 and 57.3 ± 1.7 U/mg, respectively, which are much higher
than that of wild-type D-LDH. In the D-LDH from L. bulgaricus, Leu76 and Ser235 are
located on adjacent structural elements but not close enough to interact with each other
(15.6 Å ) [17]. Therefore, this suggested that the mutations at position 75th and 234th
independently enhanced the activity of D-LDH. Double mutation at 75th and 234th
synergistically enhanced the activity of D-LDH.
3.5. Kinetic study of D-LDH
The kinetic parameters of D-LDHs were determined using sodium pyruvate as
a substrate. The kinetic parameters Km and kcat of D-LDHs are summarized in Table 1.
D-LDH_A100V, _T260V, _N263G, and _F299Y had higher Km than the wild-type
16

D-LDH, leading to decreased kcat/Km values. D-LDH_A234S had lower Km and higher
kcat, resulting in the 1.9-fold increased kcat/Km compared with that of the wild-type
D-LDH. The kcat/Km value of D-LDH_A234G increased by 1.2-fold because of the
increased kcat. From this result, the mutation at position 234 would affect rather the
catalytic efficiency than the affinity between D-LDH and pyruvate. The sequence
similarity of D-LDH from S. laevolacticus and L. bulgaricus is 80%. The residue 234 of
D-LDH from S. laevolacticus may have significantly affected the catalytic activity
because Arg236 of D-LDH, which is the active center, from L. bulgaricus corresponds
to Arg235 of D-LDH from S. laevolacticus. Most strikingly, D-LDH_T75L/A234G and
_T75L/A234S had lower Km and much higher kcat. Therefore, the kcat/Km value of
D-LDH_T75L/A234G and _T75L/A234S increased by 5.0 and 6.8-fold, respectively.
These results suggest that the replacement of Tyr75 and Ala234 synergistically improves
catalytic efficiency.
17

4. Conclusions
Ala234 of D-LDH played an important role in catalytic reaction. Site-saturation
mutagenesis indicated that the catalytic efficiency depends greatly on the size of the
side chain at position 234. Furthermore, double mutation at 75th and 234th amino acid
residues enhanced the catalytic activity. The catalytic efficiency (kcat/Km) of
D-LDH_T75L/A234S was 6.8-fold higher than that of wild-type D-LDH. In previous
researches, various characterizations of D-LDHs have been reported [18, 19]. The
previous research showed that the improvement of enzymatic efficiency of the mutated
D-LDH increased the microbial production of D-lactic acid [20]. In this study,
enhancement of the catalytic activity of D-LDH from S. laevolacticus was achieved.
This is fist report on enhancement of the catalytic activity of D-LDH. Mutated D-LDH
with high catalytic activity would contribute to the improvement of D-lactic acid
productivity in recombinant microorganism. Thus, the mutant D-LDH could be applied
for the production of D-lactic acid.
18

References
[1] R. Datta, S.P. Tsai, P. Bonsignore, S.H. Moon, J.R. Frank, Technological and
economic potential of poly( lactic acid) and lactic acid derivatives, Microbiol. Rev.
16 (1995) 221–231.
[2] K. Madhavan Nampoothiri, N.R. Nair, R.P. John, An overview of the recent
developments in polylactide (PLA) research, Bioresour. Technol. 101 (2010)
8493–8501.
[3] M.A. Abdel-Rahman, Y. Tashiro, K. Sonomoto, Recent advances in lactic acid
production by microbial fermentation processes, Biotechnol. Adv. 31 (2013)
877–902.
[4] L. Wang, Y. Cai, L. Zhu, H. Guo, B. Yu, Major role of NAD-dependent lactate
dehydrogenases in the production of L-lactic acid with high optical purity by the
thermophile Bacillus coagulans, Appl. Environ. Microbiol. 80 (2014) 7134–7141.
[5] J. Lunt, Large-scale production, properties and commercial applications of
polylactic acid polymers, Polym. Degrad. Stabil. 59 (1998) 145–152.
[6] H. Sawai, K. NA, N. Sasaki, T. Mimitsuka, S. Minegishi, M. Henmi, K. Yamada,
S. Shimizu, T. Yonehara, Membrane-integrated fermentation system for improving
19

the optical purity of D-lactic acid produced during continuous fermentation, Biosci.
Biotechnol. Biochem. 75 (2011) 2326–2332.
[7] Y. Ikada, K. Jamshidi, H. Tsuji, S.H. Hyon, Stereocomplex formation between
enantiomeric poly(lactides), Macromolecules 20 (1987) 904-906.
[8] D. Garlotta, A Literature Review of Poly(Lactic Acid), J. Polym. Environ. 9 (2001)
63–84.
[9] R.P. John, K.M. Nampoothiri, A. Pandey, Fermentative production of lactic acid
from biomass: an overview on process developments and future perspectives, Appl.
Micrrobiol. Biotechnol. 74 (2007) 524–534.
[10]Y. Wang, T. Tian, J. Zhao, J. Wang, T. Yan, L. Xu, Z. Liu, E. Garza, A. Iverson, R.
Manow, C. Finan, S. Zhou, Homofermentative production of D-lactic acid from
sucrose by a metabolically engineered Escherichia coli, Biotechnol. Lett. 34
(2012) 2069–2075.
[11] Z. Zheng, B. Sheng, C. Ma, H. Zhang, C. Gao, F. Su, P. Xu, Relative catalytic
efficiency of ldhL- and ldhD-encoded products is crucial for optical purity of lactic
acid produced by Lactobacillus strains, Appl. Environ. Microbiol. 78 (2012)
3480–3483.
20

[12] Y. Li, L. Wang, J. Ju, B. Yu, Y. Ma, Efficient production of polymer-grade
D-lactate by Sporolactobacillus laevolacticus DSM442 with agricultural waste
cottonseed as the sole nitrogen source, Bioresour. Technol. 142 (2013) 186–191.
[13] H. Sawai, K. Yamada, T. Mimitsuka, K. Sawai, T. Yonehara, Y. Ito, M. Henmi,
2009. US Patent Application, US20090269812.
[14] C. Jun, Y.S. Sa, S.A. Gu, J.C. Joo, S. Kim, K.J. Kim, Y.H. Kim, Discovery and
characterization of a thermostable d-lactate dehydrogenase from Lactobacillus
jensenii through genome mining, Process. Biochem. 48 (2013) 109-117.
[15] J. Zhang, G. Gong, X. Wang, H. Zhang, W. Tian, Positive selection on D-lactate
dehydrogenases of Lactobacillus delbrueckii subspecies bulgaricus, IET. Syst.
Biol. 9 (2015) 172–179.
[16] Y. Ishikura, S. Tsuzuki, O. Takahashi, C. Tokuda, R. Nakanishi, T. Shinoda, H.
Taguchi, Recognition site for the side chain of 2-ketoacid substrate in D-lactate
dehydrogenase, J. Biochem. 138 (2005) 741-749.
[17] A. Razeto, S. Kochhar, H. Hottinger, M. Dauter, K.S. Wilson, V.S. Lamzin,
Domain closure, substrate specificity and catalysisof D-lactate dehydrogenase from
Lactobacillus bulgaricus, J. Mol. Biol. 318 (2002) 109–119.
21

[18] N. Bernard, T. Ferain, D. Garmyn, P. Hols, J. Delcour, Cloning of the D-lactate
dehydrogenase gene from Lactobacillus delbrueckii subsp. bulgaricus by
complementation in Escherichia coli, FEBS Lett. 290 (1991) 61-64.
[19] S. J. Holton, M. Anandhakrishnan, A. Geerlof, M. Wilmanns, Structural
characterization of a D-isomer specific 2-hydroxyacid dehydrogenase from
Lactobacillus delbrueckii ssp. bulgaricus, J. Struct. Biol. 181 (2013) 179-184.
[20] H. Meng, P. Liu, H. Sun, Z. Cai, J. Zhou, J. Lin, Y. Li, Engineering a D-lactate
dehydrogenase that can superefficiently utilize NADPH and NADH as cofactors.
Sci. Rep. (2016) 6: 24887.
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Figure captions
Fig. 1. Multiple sequence alignment of D-LDH from S. laevolacticus, L. bulgaricus, and
L. casei. Amino acid sequences of D-LDHs were aligned using multiple alignment
program of the Genetyx software (Genetyx, Tokyo, Japan). The red characters show
amino acid residues with hydrophobic side chains. The triangle and asterisk indicate the
substrate binding site and active center of L. bulgaricus, respectively. The circle
represents the mutated residue.
Fig. 2 The position of the replaced amino acid residues in D-LDH from S. laevolacticus.
Ten replaced amino acid residues and active center (Arg235, Glu264, and His295) are
represented by yellow CPK models and red stick models in the solid ribbon model of
D-LDH from L. bulgaricus (PDB ID 1J49), respectively.
Fig. 3. SDS-PAGE analysis of the expressed and purified wild-type D-LDH. Lane M:
protein markers, Lane 1: soluble fraction in cells from 50 μl-culture, Lane 2: insoluble
fraction in cells from 50 μl-culture, Lane 3: purified wild-type D-LDH.
Fig. 4. Thermal stability of the wild-type D-LDH.
23

Fig. 5. The specific activities of the wild-type and various mutant D-LDHs of S.
laevolacticus. Error bars represent the standard deviations of the means for three
independent experiments.
24

S. laevolacticus 1 -MKFLMYGVQDHERATIENWANQH-QVEIATTSDFLSEDTVAQSKGFDGI 48
L. bulgaricus
L. casei
1 MTKIFAYAIREDEKPFLKEWEDAHKDVEVEYTDKLLTPETVALAKGADGV 50
1 -MKIIAYGARVDEIQYFKQWAKDT-GNTLEYHTEFLDENTVEWAKGFDGI 48
S. laevolacticus 49 CIQQPIALGGPNLYTQLKNNGIKQIATRTAGYDMIDLNEAEKNSLLVTNV 98
L. bulgaricus
L. casei
51 VVYQQLDYTA-ETLQALADNGITKMSLRNVGVDNIDMAKAKELGFQITNV 99
49 NSLQITPYAA-GVFEKMHAYGIKFLTIRNVGTDNIDMTAMKQYGIRLSNV 97
S. laevolacticus 99 PAYSPYAVAELAVTQAMQLVRHIPEFNKRVAGKDFRWSG-LISREIRSLT 147
L. bulgaricus
L. casei
100 PVYSPNAIAEHAAIQAARILRQDKAMDEKVARHDLRWAP-TIGREVRDQV 148
98 PAYSPAAIAEFALTDTLYLLRNMGKVQAQLQAGDYEKAGTFIGKELGQQT 147
S. laevolacticus 148 VGIVGTGRIGATAAQLFKGLGAKIIGFDQYPNDRLNGILDYRPSLEDVLK 197
L. bulgaricus
L. casei
149 VGVIGTGHIGQVFMQIMEGFGAKVIAYDIFRNPELEKKGYYVDSLDDLYK 198
148 VGVMGTGHIGQVAIKLFKGFGAKVIAYDPYPMKGDHPDFDY-VSLEDLFK 196
S. laevolacticus 198 EADIISLHTPLFDSTRHMINKSTLKLMKNSAYLINVARGGLIKTEDLIEA 247
L. bulgaricus
L. casei
199 QADVISLHVPDVPANVHMINDESIAKMKQDVVIVNVSRGPLVDTDAVIRG 248
197 QSDIIDLHVPGIEQNTHIINEAAFNLMKPGAIVINTARPNLIDTQAMLSN 246
＊
S. laevolacticus 248 LENGEIAGAALDTFENELMINKDLSKQ-PLNDPLLSKLLDMEQVLLTPHV 296
L. bulgaricus
L. casei
249 LDSGKIFGYAMDVYEGEVGIFNEDWEGKEFPDARLADLIARPNVLVTPHT 298
247 LKSGKLAGVGIDTYEYETEDLLNLAKHGSFKDPLWDELLGMPNVVLSPHI 296
＊
＊
S. laevolacticus 297 GFFTETAIQNIVEGALDSVVEVLKTGTSKNLVQAQPLSAK
336
333
333
L. bulgaricus
L. casei
299 AFYTTHAVRNMVVKAFDNNLELVEGKEAETPVKVG-----
297 AYYTETAVHNMVYFSLQHLVDFLTNGETSTEVTGPAK---
Fig. 1
25

Fig. 2
26

Fig. 3
27

Fig. 4
Fig. 5
28

Tables
Table 1:Kinetics parameters of the purified His-tagged D-LDHs.
D-LDH
kcat×10−4(s−1)
4.62
Km (mM)
14.8
14.1
9.20
67.3
9.49
21.4
136
kcat/Km×10−6 (M−1s−1)
D-LDH (wild-type)
D-LDH_T75L
3.12
5.51
3.38
0.236
5.99
2.65
0.530
3.11
2.10
3.70
1.26
0.575
21.1
15.7
7.77
D-LDH_A78V
D-LDH_A100V
D-LDH_A234S
D-LDH_T260V
D-LDH_N263G
D-LDH_G297A
D-LDH_F299Y
D-LDH_A234G
D-LDH_A234C
D-LDH_A234T
D-LDH_T75L/A234S
D-LDH_T75L/A234G
3.11
1.59
5.68
5.66
7.21
1.90
6.10
20.8
18.5
5.98
15.3
10.6
11.5
4.37
6.85
0.755
0.880
22.5
18.1
29